Vpliv degradacije elektrod med elektroporacijo na procese celjenja celične membrane

magistrsko delo

Tina Turk (Author), Alenka Maček Lebar (Mentor)

Abstract

Elektroporacija je proces, pri katerem se zaradi izpostavitve celic ali tkiva visokonapetostnim električnim pulzom začasno poveča prepustnost celične membrane, kar omogoča prehajanje različnih ionov in molekul. Kot uveljavljen osnovni mehanizem elektroporacije prevladuje prerazporeditev lipidnih molekul celične membrane v prevodne poti, t.i. pore. Poleg tvorbe por raziskave zadnjih let potrjujejo, da izpostavitev pulzirajočim električnim poljem povzroča tudi kemijske spremembe v celici in vpliva na delovanje membranskih proteinov, kar lahko dodatno poveča prepustnost celične membrane. Med elektroporacijo z elektrodami iz različnih kovin se biološki vzorec segreva, na stiku med elektrodami in medijem pa potekajo elektrokemijske reakcije. Pomemben elektrokemijski proces je oksidacija kovine elektrod, zaradi česar se v biološki vzorec sproščajo kovinski ioni. Pokazano je bilo, da predvsem izločanje železovih ionov po izpostavitvi električnim pulzom sproži procese, ki lahko povzročijo citotoksičnost medija in vplivajo na preživetje celic v biološkem vzorcu. Elektroporacija lahko poteka reverzibilno, pri čemer se celična membrana postopoma obnovi. V kolikor je povečanje prepustnosti celične membrane preveliko, pride do zmanjšane zmožnosti popravljanja poškodb in celične smrti, kar imenujemo ireverzibilna elektroporacija. Procesi celjenja celične membrane vključujejo izločanje različnih snovi neposredno skozi polprepustno celično membrano ali pa v zunajceličnih veziklih. Namen naše raziskave je bil ugotoviti kako elektrode iz različnih kovin vplivajo na celične procese po elektroporaciji. Osredotočili smo se na vlogo inkubacijskega medija pri okrevanju celic po elektroporaciji z elektrodami iz nerjavečega jekla in aluminija in njegov vpliv na preživetje celic iz sveže celične kulture. Raziskovali smo kako prisotnost ionov Fe2+ v rastnem mediju med elektroporacijo z elektrodami iz aluminija vpliva na citotoksičnost inkubacijskega medija in opazovali tvorbo mehurjev na celični membrani po izpostavitvi električnemu polju z obema vrstama elektrod. Vse poskuse smo izvedli s celično linijo pridobljeno iz ovarijev kitajskih hrčkov (CHO). Rezultati so pokazali, da je inkubacijski medij po elektroporaciji z elektrodami iz nerjavečega jekla povzročil smrt celotne populacije celic iz sveže celične kulture, medtem ko je bilo preživetje celic iz sveže celične kulture pri uporabi elektrod iz aluminija primerljivo s kontrolo, kar pomeni, da material elektrod vpliva na celične procese po elektroporaciji. Dodana koncentracija ionov Fe2+ pri elektroporaciji z elektrodami iz aluminija, ki je povzročila statistično značilno zmanjšanje deleža metabolno aktivnih celic iz sveže celične kulture v inkubacijskem mediju, je bila 5 mM. To je nekoliko višja vrednost od pričakovane in nakazuje na to, da so v celične procese, ki povzročajo citotoksičnost inkubacijskega medija po elektroporaciji vpleteni tudi drugi dejavniki, med katere sodi izločanje železovih ionov Fe3+. Pri opazovanju nastajanja mehurjev na celični membrani smo ugotovili, da elektroporacija z elektrodami iz nerjavečega jekla povzroča izrazitejše strukturne spremembe celične membrane. Po elektroporaciji z elektrodami iz aluminija do podobnih učinkov pride pri višjih jakostih električnega polja.

Keywords

elektroporacija;železovi ioni;inkubacijski medij;citotoksičnost;celična membrana;nastajanje mehurjev;magisteriji;

Data

Language:	Slovenian
Year of publishing:	2023
Typology:	2.09 - Master's Thesis
Organization:	UL FE - Faculty of Electrical Engineering
Publisher:	[T. Turk]
UDC:	602.621(043.3)
COBISS:	165359875
Views:	25
Downloads:	1
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Effect of electrode degradation during electroporation on cell membrane recovery processes
Secondary abstract:	Electroporation is a process in which the exposure of cells or tissue to high-voltage electric pulses temporarily increases the permeability of the cell membrane, allowing the passage of various ions and molecules. The prevailing fundamental mechanism of electroporation involves the redistribution of lipid molecules in the cell membrane to form conductive pathways, known as pores. In addition to pore formation, recent research confirms that exposure to pulsating electric fields also induces chemical changes within the cell and affects the function of membrane proteins, further enhancing membrane permeability. During electroporation with electrodes made of different metals, the biological sample heats up, and electrochemical reactions occur at the interface between the electrodes and the medium. An important electrochemical process involves the oxidation of the electrode metal, leading to the release of metal ions into the biological sample. It has been shown that the release of iron ions after exposure to electric pulses, triggers processes that can lead to cytotoxicity of the medium and influence cell survival in the biological sample. Electroporation can occur reversibly, with the cell membrane gradually recovering. However, if the increase in membrane permeability is too high, it can lead to reduced ability to repair damage and cell death, a condition referred to as irreversible electroporation. The processes involved in cell membrane resealing include the release of various substances either directly through the partially permeable cell membrane or through extracellular vesicles. The purpose of our study was to determine how electrodes made of different metals affect cellular processes after electroporation. We focused on the role of the incubation medium in the recovery of cells following electroporation with stainless steel and aluminum electrodes and its impact on the viability of cells from a fresh cell culture. We investigated how the presence of Fe2+ ions in the growth medium during electroporation with aluminum electrodes influenced the cytotoxicity of the incubation medium and observed cell membrane bleb formation after exposure to an electric field with both types of electrodes. In all experiments, Chinese hamster ovary (CHO) cells were used. The results showed that the incubation medium after electroporation with stainless steel electrodes resulted in the death of the entire population of cells from the fresh cell culture, whereas the survival of cells from the fresh cell culture when using aluminum electrodes was comparable to the control. This suggests that the electrode material influences cellular processes post-electroporation. The added concentration of Fe2+ ions during electroporation with aluminum electrodes, which led to a statistically significant reduction in the proportion of metabolically active cells from the fresh cell culture in the incubation medium, was 5 mM. This value is slightly higher than expected and indicates that other factors, including the release of Fe3+ ions, are involved in the cellular processes that cause cytotoxicity in the incubation medium after electroporation. While observing cell membrane blebbing, it was found that electroporation with stainless steel electrodes causes more pronounced structural changes in the cell membrane. Similar effects occur with aluminum electrodes at higher electric field strengths.
Secondary keywords:	electroporation;iron ions;incubation medium;cytotoxicity;cell membrane;bleb formation;
Type (COBISS):	Master's thesis/paper
Study programme:	1000316
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Fak. za elektrotehniko
Pages:	VI, 52 str.
ID:	19921146